To date very limited knowledge is available on 3-ketosteroid 9α-hydroxylase (KSH), the enzyme performing the 9α-hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in microbial sterol/steroid degradation. No nucleotide sequences of the genes encoding KSH components have been reported. Furthermore, difficulties are faced during enzyme purification procedures (Chang, F. N. et al. Biochemistry (1964) 3:1551-1557; Strijewski, A. Eur. J. Biochem. (1982) 128:125-135). A three-component monooxygenase with KSH activity has been partially purified from Nocardia sp. M117 and was found to constitute a three-component enzyme system, composed of a flavoprotein reductase and two ferredoxin proteins (Strijewski, A. Eur. J. Biochem. (1982) 128:125-135). In Arthrobacter oxydans 317, 9α-hydroxylation of the steroid poly-cyclic ring structure appeared plasmid-borne (Dutta, R. K. et al. J. Basic Microbiol. (1992) 32:317-324). Nucleotide sequence analysis of the plasmid, however, was not reported.
The lack of genetic data has hampered the construction of molecularly defined mutant strains with desired properties (i.e. blocked 9α-hydroxylation of steroids) by genetic engineering. Mutants have been isolated by classical mutagenesis, but these strains usually are inadequate in industrial processes mostly due to genetic instability and/or low bioconversion efficiencies. Molecularly defined mutants have advantages compared to mutants generated by classical mutagenesis. The constructed mutants are genetically stable and the introduced mutations are well-defined genetic modifications. Construction of genetically engineered strains make the widespread use of chemical agents to block 9α-hydroxylation (e.g. α,α-dipyridyl, o-phenanthroline) obsolete. Chemical agents used to block KSH activity mostly are not reaction specific and inhibit other important enzymatic reactions (e.g. sterol 26-hydroxylation in sterol side chain degradation), which may have negative effects on sterol bioconversion efficiency. The use of defined mutants by genetic engineering overcomes these problems.
3-Ketosteroid 9α-hydroxylase (KSH) is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the conversion of AD into 9α-hydroxy-4-androstene-3,17-dione (9OHAD) and ADD into the chemically unstable compound [9OHADD]. KSH activity has been found in many bacterial genera (Martin, C. K. A. Adv. Appl. Microbiol. (1977) 22: 29-58; Kieslich, K. J Basic Microbiol. (1985) 25: 461-474; Mahato, S. B. et al. Steroids (1997) 62: 332-345): e.g. Rhodococcus (Datcheva, V. K. et al. Steroids (1989) 54:271-286; Van der Geize et al. FEMS Microbiol. Lett. (2001) 205: 197-202, Nocardia (Strijewski, A. Eur. J. Biochem. (1982) 128:125-135), Arthrobacter (Dutta, R. K. et al. J. Basic Microbiol. (1992) 32:317-324) and Mycobacterium (Wovcha, M. G. et al. Biochim Biophys Acta (1978) 531:308-321). Bacterial strains lacking KSH activity are being considered important in sterol/steroid biotransformation. Mutants blocked in KSH activity will be able to perform only the KSTD (3-ketosteroid Δ1-dehydrogenase) reaction, thereby allowing selective Δ1-dehydrogenation of steroid compounds. Examples are the cortisol biotransformation into prednisolone and the AD biotransformation into ADD. Sterol bioconversion by mutants blocked at the level of steroid 9α-hydroxylation may also carry out a selective degradation of the sterol side chain thereby accumulating AD and/or ADD which are excellent precursors for the synthesis of bioactive steroid hormones.
According to one aspect of the present invention, the isolated polynucleotide sequences of two genes, designated kshA and kshB of Rhodococcus erythropolis are now provided: SEQ ID NO:1 and SEQ ID NO:2, respectively. KshA protein is encoded by nucleotides 499-1695 of SEQ ID NO:1 and KshB protein by nucleotides 387-1427 of SEQ ID NO:2. Thus, in particular preferred are polynucleotides comprising the complete coding DNA sequences of the nucleotides 499-1695 of SEQ ID NO:1 and of the nucleotides 387-1427 of SEQ ID NO:2, respectively.
Furthermore, to accommodate codon variability the invention also includes sequences coding for the same amino acid sequences of the KshA protein and the KshB protein. Also portions of the coding sequences coding for individual domains of the expressed protein are part of the invention as well as allelic and species variations thereof. Sometimes, a gene is expressed as a splicing variant, resulting in the inclusion of an additional exon sequence, or the exclusion of an exon. Also a partial exon sequence may be included or excluded. A gene may also be transcribed from alternative promotors that are located at different positions within a gene, resulting in transcripts with different 5′ ends. Transcription may also terminate at different sites, resulting in different 3′ ends of the transcript. These sequences as well as the proteins encoded by these sequences all are expected to perform the same or similar functions and form also part of the invention. The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequence disclosed herein can be readily used to isolate the complete genes which in turn can easily be subjected to further sequence analyses thereby identifying sequencing errors.
The present invention further relates to polynucleotides having slight variations or having polymorphic sites. Polynucleotides having slight variations encode polypeptides which retain the same biological function or activity as the natural, mature protein.